Interleukin-8 (IL-8), neutrophil-activating peptide 2 (NAP-2), and gro /melanoma growth-stimulatory activity (GRO) are structurally related human peptides that act as mediators of inflammation. Two human neutrophil IL-8 receptors have been cloned, type A (IL8RA) and type B (IL8RB). The two receptors bind IL-8 with high affinity, but IL8RB also binds GRO and NAP-2. Our immediate goal is to define the regions to which IL-8, GRO, and NAP-2 bind the IL8RB. Using the mammalian expression vector pcDNA 3 (Invitrogen) we have cloned and expressed the IL8RB into the human cell line 293 (human kidney epithelial). The sequence of the IL8RB fragment was confirmed by the dideoxy sequencing method. Stable transfection has been accomplished by selection with the antibiotic G418 and has been confirmed by fluorescence activated cell sorting (FACS) and radioactive ligand binding studies with IL8. Saturable binding is seen at approximately 2 nM IL8. Working with Dr. Ernesto DeNardin, I'm currently characterizing binding of the ligands IL-8, NAP-2 and GRO-alpha to the IL8RB. This work involves radioactive binding assays with peptide inhibition to map the binding regions of the receptor. I have synthesized eight peptides, approximately 20 amino acids in length, that correspond to the extracellular regions of the IL8RB. These peptides have been purified by HPLC and composition confirmed by amino acid analysis. Preliminary data suggests that the N-terminal region of the receptor and he first extracellular loop are critical areas for ligand binding. Peptides corresponding to these regions demonstrate approximately 50% inhibition of control binding levels. These studies will provide information on the function of the IL8RB and may allow for the future design of receptor antagonists. Receptor antagonists may provide a novel means of modulating destructive inflammatory reactions, including those seen in periodontal disease.